Effector CLas0185 targets methionine sulphoxide reductase B1 of Citrus sinensis to promote multiplication of ‘Candidatus Liberibacter asiaticus’ via enhancing enzymatic activity of ascorbate peroxidase 1

Abstract Citrus huanglongbing (HLB) has been causing enormous damage to the global citrus industry. As the main causal agent, ‘Candidatus Liberibacter asiaticus’ (CLas) delivers a set of effectors to modulate host responses, while the modes of action adopted remain largely unclear. Here, we demonstrated that CLIBASIA_00185 (CLas0185) could attenuate reactive oxygen species (ROS)‐mediated cell death in Nicotiana benthamiana. Transgenic expression of CLas0185 in Citrus sinensis ‘Wanjincheng’ enhanced plant susceptibility to CLas. We found that methionine sulphoxide reductase B1 (CsMsrB1) was targeted by the effector, and its abundance was elevated in CLas0185‐transgenic citrus plants. Their interaction promoted CLas proliferation. We then determined that CsMsrB1 sustained redox state and enzymatic activity of ascorbate peroxidase 1 (CsAPX1) under oxidative stress. The latter reduced H2O2 accumulation and was associated with host susceptibility to CLas infection. Consistently, citrus plants expressing CLas0185 and CsMsrB1 conferred enhanced APX activity and decreased H2O2 content. Taken together, these findings revealed how CLas0185 benefits CLas colonization by targeting CsMsrB1, which facilitated the antioxidant activity and depressed ROS during pathogen infection.

Secreted proteins of pathogens, called effectors, are prominent players to aid infection by manipulating plant immunity, which creates favourable environments for pathogen colonization and proliferation (Jones & Dangl, 2006).Genome sequence analysis of CLas revealed its ability to deliver effectors into host cells using a general Sec-dependent secretion apparatus (Duan et al., 2009).Prasad et al. (2016) characterized 86 Sec-dependent CLas effectors, and many of them have been identified according to their functions on plant immune responses.For instance, CLas4425 induces cell death in Nicotiana benthamiana and enhances citrus susceptibility to CLas by interfering with salicylic acid-mediated plant immunity (Zhang, Wang, et al., 2023).Phylogenetic analysis indicated that effectors are highly conserved between CLas isolates but not across all three HLB species (Thapa et al., 2020).
The inability of the HLB pathogens to be cultured in vitro and the time-consuming generation of transgenic citrus plants restrict the dissection of HLB pathogenicity.The molecular mechanisms of a few CLas effectors in Citrus-CLas interactions have been discovered: SDE1 (CLas Sec-delivered effector 1) inhibits immune protease activity to suppress citrus defence (Clark et al., 2018).SDE15 suppression of plant immunity is dependent on accelerated cell death 2 (ACD2) (Pang et al., 2020).SDE3 and SDE4405 promote pathogen infection via manipulating host autophagy (Shi, Gong, et al., 2023;Shi, Yang, et al., 2023).A prophage-encoded effector, AGH17488, targets ascorbate peroxidase 6 (CsAPX6) to scavenge reactive oxygen species (ROS) accumulation (Du et al., 2023).However, it is far from sufficient to fully elucidate how CLas effectors contribute to HLB development.
In plants, physiological responses to environmental constraints disrupt internal balance, among which redox homeostasis is largely manipulated during plant-pathogen interactions (Bleau & Spoel, 2021;Camejo et al., 2016).ROS are a group of molecules derived from O 2 , consisting of hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH − ), and singlet oxygen ( 1 O 2 ), which serve as a crucial component in redox homeostasis, and contribute to plant immunity (Mittler, 2017).Maintaining a basal level of ROS is necessary for life, while excessive ROS are toxic and cause oxidative stress.
Thereby plants confer a complex array of systems to neutralize the effect, such as antioxidant system (Caverzan et al., 2012).This system comprises enzymatic antioxidants and some low molecular mass antioxidants.Among the enzymes, APX family proteins have a high affinity for H 2 O 2 and contribute greatly to H 2 O 2 detoxification (Sofo et al., 2015).They are involved in physiological responses, such as leaf senescence, and programmed cell death (de Pinto et al., 2013;Ribeiro et al., 2017).Besides the antioxidant system, the repair system is also associated with redox homeostasis, in which methionine sulphoxide reductase (Msr) family proteins are the main player (Rey & Tarrago, 2018).
CLas0185, delivered via the Sec translocon, comprises an Nterminal signal peptide with 19 amino acids (aa) and the mature form with 49 aa (Prasad et al., 2016).Due to its high level of amino acid sequence identity among CLas strains, CLas0185 was predicted to be a core effector (Thapa et al., 2020).Using transient expression and genetic approaches, we identified that CLas0185 manipulates plant defence responses and contributes to HLB progression.Screening for the targets in citrus plants, we confirmed the interaction between CLas0185 and CsMsrB1.Using Agrobacterium rhizogenesmediated hairy root transformation in a CLas0185-transgenic Citrus sinensis background, we determined that CLas0185 contribution to HLB pathogenicity is dependent on CsMsrB1.We further verified the association between CsMsrB1 and CsAPX1, and the latter was susceptible for HLB.Here, we conducted a time-saving approach to analyse the role of effector-interacting protein in HLB development with hairy root transformation.Moreover, the study provided a detailed examination of the molecular events associated with the depression of plant defence responses in CLas-accumulated tissues.

| CLas0185 suppresses ROS-mediated cell death in N. benthamiana
Based on the Sec-dependent CLas effectors identified by Prasad et al. (2016), we employed a functional screen using potato virus X (PVX) vector pGR107 to select potential virulence factors.In this assay, pro-apoptotic mouse protein BCL2-associated X protein (BAX) and green fluorescent protein (GFP) served as the positive and negative controls, respectively.The results indicated that CLas0185 attenuates the BAX-induced ROS burst and cell death (Figure 1a).Intriguingly, it could also block the cell death-inducing activity of CLas04425 (Figure 1b), which was previously determined to trigger cell death in N. benthamiana (Zhang, Wang, et al., 2023).Immunoblot verified that all the recombinant proteins were properly expressed at the expected size (Figure 1).

| CLas0185 enhances plant susceptibility to CLas
The expression profiles of CLas0185 were analysed to investigate its role in CLas virulence.The results showed that CLas0185 was highly expressed (c. 17-fold) in infected citrus plants compared to that in psyllids (Figure 2a).Within different citrus tissues, it was largely expressed in rootlets (Figure 2b).According to the importance of roots in CLas early infection (Johnson et al., 2014), our data imply that CLas0185 could have an effect on the initiation of pathogen colonization.
To determine the contribution of CLas0185 to disease progression, CLas0185-transgenic citrus plants (0185-OE) were generated via Agrobacterium-mediated transformation, which was confirmed with PCR and β-glucuronidase (GUS) assays (Figure S1a-c).Three independent 0185-OE lines were obtained, and the transcript levels of CLas0185 were detected with reverse transcription-quantitative PCR (RT-qPCR) (Figure S1d).They showed no difference in growth phenotype compared with wild-type (WT) plants (Figure S1e,f).
Citrus plants were graft-inoculated using stems from diseased trees.
A significant increase of CLas titres was detected in the 0185-OE group compared to the WT group at 2 and 3 months post-inoculation (mpi) (Figure 2c).Hence, CLas0185 accelerated pathogen multiplication during the early stages of infection.
As elicitor-triggered ROS burst was inhibited in N. benthamiana expressing CLas0185, we examined H 2 O 2 accumulation in leaves of 0185-OEs.Before infection, H 2 O 2 content was depressed in 0185-OEs, indicating that the effector compromised citrus basal immunity (Figure 2d).At 6 mpi, H 2 O 2 levels were elevated in the WT group compared to 0185-OEs.We further monitored the transcript levels of plant defence-related genes in citrus leaves, including pathogenesis-related genes (CsPRs) and phloem protein 2 (CsPP2) within 4 mpi.We found their expression was markedly depressed in 0185-OEs at 1 mpi compared with that in the WT group (Figure 2eh).Of interest, HLB symptoms observed at 12 mpi were attenuated in 0185-OEs (Figure 2i).Taken together, our findings demonstrate that CLas0185 enhances citrus susceptibility to CLas.

| CLas0185 contribution to HLB pathogenicity is dependent on CsMsrB1
To understand the potential role of CLas0185 in citrus, we performed a yeast two-hybrid (Y2H) assay using a C. sinensis cDNA library to identify candidate CLas0185-interacting proteins (Table S1).
Among them, the C. sinensis protein annotated as CsMsrB1 (NCBI accession XP_006477977.2) is implicated in redox homeostasis (Rey & Tarrago, 2018).It has been reported that Msr proteins play roles in plant response to biotic stresses (Fu et al., 2023;Gao et al., 2012); however, little is known about the underlying mechanisms.Hence, we chose CsMsrB1 for further study.As MsrB1 belongs to the Msr family, a phylogenetic tree of Msr proteins was constructed from Nicotiana tabacum (10), Zea mays (3), Musa acuminata (6), and C. sinensis (6).Msr proteins in citrus are divided into two subfamilies; CsMsrB1 is in Cluster VI belonging to MsrB subgroup (Figure S2).
The MsrB subfamily proteins possess a conserved SelR domain, which plays a role in maintaining intracellular redox homeostasis via reducing methionine sulphoxide to methionine (Figure S2).
To validate the interaction of CLas0185 and CsMsrB1, a pairwise Y2H assay was performed.All yeast transformants grew on control plates.Nevertheless, only the transformants containing CLas0185 and CsMsrB1 could induce β-galactosidase activity and exhibited a positive signal on stringent SD/−Leu/−Trp/−His/−Ade medium (Figure 3a).We next conducted a luciferase complementation imaging (LCI) assay in N. benthamiana to validate the interaction in vivo.
In this assay, luciferase complementation indicated that CLas0185 bound with CsMsrB1, observed with chemiluminescence apparatus at 3 dpi (Figure 3b).We further used a pull-down assay and confirmed the interaction between CLas0185 and CsMsrB1 in vitro (Figure 3c).
F I G U R E 1 CLIBASIA_00185 (CLas0185) attenuates hypersensitive cell death in Nicotiana benthamiana.CLas0185 blocks (a) pro-apoptotic mouse protein BCL2-associated X protein (BAX)-induced cell death and (b) CLas4425-induced cell death.Leaves were infiltrated with buffer or Agrobacterium tumefaciens cells expressing CLas0185 or green fluorescent protein (GFP), either alone or followed 24 h later with A. tumefaciens cells harbouring BAX or CLas4425.The circles indicate infiltration sites.To visualize the reactive oxygen species burst, leaves were detached at 3 days post-inoculation (dpi) for 3,3′-diaminobenzidine (DAB) staining.The leaves for programmed cell death observation were photographed at 5 dpi after trypan blue staining.For symptom observation, leaves were collected and photographed at 7 dpi.Anti-HA and anti-FLAG antibodies were used to detect the expression of the indicated constructs at 3 dpi, and equal loading of each sample was confirmed by immunoblotting of RuBisCO.All experiments were performed in triplicate.The numbers in each circled area indicate summary data representing the number of infiltrated area(s) exhibiting hypersensitive responses over the total number of leaf areas infiltrated with a particular construct or combination of constructs.
We then analysed the abundance of CsMsrB1 to see if it could be affected by the effector.CsMsrB1-FLAG and GUS-FLAG were transiently expressed in leaves of either 0185-OE or WT group, and total proteins were extracted at 3 dpi.The abundance of CsMsrB1-FLAG was notably higher in transgenic plants than in WT lines, while no significant difference was observed between 0185-OEs and WTs in the expression of GUS-FLAG (Figure 3d).The results indicated that CLas0185 increased the abundance of CsMsrB1 in C. sinensis.
To explore whether CsMsrB1 is involved in the CLas0185mediated promotion of CLas infection, we generated CsMsrB1-overexpressing (CsMsrB1-OE) and CsMsrB1-silenced (CsMsrB1-RNAi) transgenic citrus hairy roots using Agrobacterium rhizogenes-mediated transformation, which developed from CLasinfected 0185-OE stem sections.The derived rootlets were verified with PCR at 3 mpi.Four CsMsrB1-OE and CsMsrB1-RNAi lines were obtained, and the overexpression/silencing efficiency was determined using RT-qPCR (Figure S3).The control groups were the rootlets derived from CLas-infected 0185-OE stem sections without transformation.In 0185-OE + CsMsrB1-OEs, CLas titres were increased while H 2 O 2 content was reduced; in 0185-OE + CsMsrB1-RNAi lines, CLas titres were decreased while H 2 O 2 content was elevated (Figure 3e-g).The results indicated that CsMsrB1 contributes to CLas0185-facilitated HLB development via inhibiting ROS accumulation.

| CsMsrB1 regulates redox state and activity of CsAPX1
Because CsMsrB1 is a prominent player in redox homeostasis, we Previous studies verified the interplay between Msr proteins and enzymatic antioxidants (Cui et al., 2022;Xiao et al., 2021).
Because CsAPX6 was targeted by another CLas effector, AGH17488 (Du et al., 2023), we focused on the interaction between CsMsrB1 and CsAPX1, which was further validated with LCI and pull-down assays (Figure 4c,d).A phylogenetic tree of APXs was constructed from C. sinensis (6), N. tabacum (3), Arabidopsis (6), and Z. mays (7), indicating a high degree of amino acid homology between CsAPX1 and AtAPX1 (Figure S6).Alignment of amino acid sequences of CsAPXs was analysed with the percentage sequence identities from 11.24% to 77.35% (Figure S6).CsMsrB1 did not interact with CsAPXs in a pairwise Y2H assay apart from with CsAPX1 (Figure S6).Using a competitive pull-down assay, we clarified that the binding between CsMsrB1 and CsAPX1 was not weakened by CLas0185 (Figure 4e).4g).Enzymatic analysis revealed that oxidization hindered CsAPX1 activity, which could be partially restored by CsMsrB1 (Figure 4h).
Homologous overexpression and VIGS assays were conducted to manipulate CsAPX1 expression in C. sinensis.After verification of protein expression at 3 dpi and the fragment insertion at 1 mpi, the overexpressing/silencing efficiency was determined using RT-qPCR (Figure S7).We then examined APX activity and H 2 O 2 in citrus plants.
Overexpression of CsAPX1 significantly elevated APX activity and reduced the H 2 O 2 level in planta, whereas silencing it decreased APX activity and increased H 2 O 2 accumulation (Figure 4h,i).The CsAPX1 expression in 2-year-old 0185-OEs was determined with RT-qPCR, indicating that it was expressed in rootlets, leaves, and midribs.
CsAPX1 expression in rootlets was lower than in leaves and midribs (Figure 4k).From our findings, we speculate that CsMsrB1 eliminates ROS by sustaining CsAPX1 activity.

| CsAPX1 is a susceptibility gene for HLB
To determine the role of CsAPX1 in CLas infection, we generated CsAPX1-OE and CsAPX1-RNAi transgenic citrus hairy roots, which were developed from CLas-infected stem sections.Using PCR and GUS staining at 3 mpi, four CsAPX1-OE and CsAPX1-RNAi lines were confirmed, and the overexpressing/silencing efficiency was determined using RT-qPCR (Figure S8).Rootlets derived from CLasinfected stem sections without transformation served as control groups.Determined with TaqMan qPCR, CLas titres were increased in the CsAPX1-OE group, while decreased in the CsAPX1-RNAi group (Figure 5a,b).The results imply that CsAPX1 serves as a susceptibility gene in response to CLas infection.Notably, APX activity was enhanced in citrus plants overexpressing CLas0185, and not changed by CLas infection (Figure 5c).Overexpression of CsMsrB1 elevated APX activity in citrus plants, as well as in CLas0185-OE rootlets, while CsMsrB1-silencing decreased its activity in both backgrounds (Figure 5d,e).We propose a working model that the effector promotes HLB development by inhibiting ROS-mediated plant immunity (Figure 5f).ROS generation increases in HLB-diseased citrus plants.CLas0185 is secreted in CLas-accumulated sieve cells, which enhances CsMsrB1 abundance, resulting in the reduction of ROS accumulation via elevating APX activity.

| DISCUSS ION
Prior studies revealed that CLas infects root during the initiation of colonization, and mainly accumulates in sieve cells where plant immune responses are inhibited (Bernardini et al., 2022;Johnson et al., 2014).Therefore, research on the attempts of the pathogen to bypass plant defence is necessary for the development of effective management strategies.In this study, we identified a CLas effector, CLas0185, that is highly expressed in rootlets and suppresses plant defence responses.Using the effector as a probe, we determined CsMsrB1 to be a target, which decreases H 2 O 2 accumulation by enhancing enzymatic antioxidant activity, thereby offering mechanistic insights into the virulence strategies taken by CLas0185 to depress citrus innate immunity and promote HLB progression.
Redox homeostasis is associated with ageing and protein functions.Its dysregulation causes severe human diseases (Lee et al., 2017;Reiterer et al., 2019).Methionine (Met) belongs to the group of amino acids most susceptible to oxidation, and a diastereomeric mixture of Met-S-O and Met-R-O is formed in this reaction, leading to dysfunctional proteins (Le et al., 2018;Vogt, 1995).They can be reversed by two distinct Msr subfamilies, namely MsrA and MsrB, corresponding to different forms of Met-O.Despite displaying a similar physiological function, the two subfamily proteins do not share homology at the primary sequence level or at the structural level.Due to the protective role in repairing oxidized proteins, Msr family proteins have been well studied in medicine (Moskovitz, 2005;Reiterer et al., 2019).The role of Msr proteins in plant physiology is receiving increasing attention, and progress has been made to dissect their functions in response to abiotic stresses.For instance, Arabidopsis root-abundant MsrB genes are involved in tolerance to oxidative stress (Li et al., 2012).Ectopic expression of ZmMsrB1 from Z. mays enhances salinity stress tolerance in Arabidopsis (Wang  et al., 2022).By contrast, Msr functions implicated in biotic stresses are more complicated.The expression of Msr genes is manipulated in response to pathogen infection (Sadanandom et al., 2000).A pepper (Capsicum annuum) CaMsrB2 gene activates defence against pathogen attack (Oh et al., 2010).MsrB8 is required for defence against avirulent pathogens in Arabidopsis (Roy & Nandi, 2017).SlMsrB5 contributes to tomato fruit defence response against Botrytis cinerea induced by methyl jasmonate (Fu et al., 2023).Notably, NIa-pro of papaya ringspot virus interacts with papaya PaMsrB1 to scavenge ROS caused by virus infection (Gao et al., 2012).Similarly, CLas0185 binding with CsMsrB1 inhibits H 2 O 2 accumulation, and their interaction contributes to CLas proliferation.As Msr family proteins fulfil key signalling roles in the transmission of ROS-related information (Chu et al., 2016;Jacques et al., 2015;Sun et al., 2016), further exploration could focus on the effect of CLas0185-CsMsrB1 interaction on ROS-mediated signalling transduction.
Previous studies verified the interplay between Msr proteins and enzymatic antioxidants.For instance, APX1 activity can be enhanced by MsrB2, which regulates the redox state of banana fruit during ripening and senescence (Xiao et al., 2021).Among these antioxidants, APX has the highest affinity for H 2 O 2 (Huang et al., 2018).It has been determined that APX expression and protein activity are manipulated in response to pathogen infection (Pérez-Clemente et al., 2014;Zhang, Song, et al., 2023), while the reduction of APX activity results in ROS accumulation, thereby enhancing plant innate immunity (Chandrashekar & Umesha, 2014;Fujiwara et al., 2016).A prior study reported that a prophage-encoded effector from CLas targets CsAPX6 to facilitate Xanthomonas citri subsp.citri infection (Du et al., 2023).Here, CsAPX1 was determined to promote HLB.These results suggest that APX plays an important role in HLB pathogenesis.In this study, we found that the expression of the CLas effector CLas0185 and its host target CsMsrB1 can enhance APX activity and inhibit ROS accumulation.It has been established that ROS accumulation intensifies HLB symptoms, which can be mitigated through foliar sprays of antioxidants (Clark et al., 2020;Ma, Pang, et al., 2022).HLB symptoms were eliminated in 0185-transgenic C. sinensis.Intriguingly, we determined that the reduction of ROS accumulation by CLas0185 contributes to CLas proliferation.Nevertheless, a decrease in bacterial titres is observed with this foliar application (Ma, Pang, et al., 2022).Hence, we suppose that CLas0185 uses multiple tactics to facilitate CLas pathogenicity.
Screened by Y2H assay, 48 candidate proteins were identified to interact with CLas0185, revealing a wide range of functions for the effector.Among them, protein disulphide-isomerase plays a role in catalysing misfolded proteins, which contributes to plant resistance (Li et al., 2020).Histone acetylation confers a function on acetylated lysine residues of the core histones, facilitating plant adaption to different stresses (Kumar et al., 2021).Notably, candidates including heat shock 70 kDa protein, small ubiquitin-related modifier, and constitutive photomorphogenesis protein regulate protein degradation (Berka et al., 2022;Ghimire et al., 2020;Ouyang & Frucht, 2021).In this study, we discovered that the amount of CsMsrB1 is enhanced in CLas0185-transgenic C. sinensis, supposing that the CLas effector could block the degradation pathway of CsMsrB1.Further studies are needed to investigate the potential roles of CLas0185.
CLas4425 has been identified as an elicitor, and its cell deathinducing activity is dependent on Botrytis-induced kinase 1 in N. benthamiana, which is a key player in ROS-mediated plant immunity (Zhang, Wang, et al., 2023).Intriguingly, CLas4425-triggered cell death can be suppressed by CLas0185.Further analysis on this countereffect would provide a growing understanding of coordinated effector activities.
To sum up, through characterization of a CLas effector,

| E XPERIMENTAL PROCEDURE S
All recombinant vectors were constructed using ClonExpress II One Step Cloning Kit (Vazyme) and were confirmed by Sanger sequencing.The strains and vectors are in Table S2; the primers used in the study are listed in Table S3.The origin of CLas strain was from Guangxi Province of China.

| Transient expression in planta
For transient expression in N. benthamiana, Agrobacterium tumefaciens GV3101 (pJIC SA_Rep) was used.BAX and CLas effector CLas4425 (Li et al., 2015;Zhang, Wang, et al., 2023) served as positive controls that induce cell death.To determine the effect of CLas0185 on ETI, N. benthamiana leaves were first infiltrated with recombinant strains of A. tumefaciens carrying CLas0185 or GFP, and the elicitor was injected in the same regions at 1 dpi.The leaves were stained using 3,3′-diaminobenzisine (DAB) staining at 3 dpi and trypan blue staining at 5 dpi (Li et al., 2019), and symptoms were observed and photographed at 7 dpi.The experiments were repeated three times.
For transient expression in C. sinensis, A. tumefaciens EHA105 was used.The suspension expressing target genes and GUS (as control) were injected in different sides of the same leaf on opposite sides of the main vein (Du et al., 2022).The experiments were repeated twice.

| Immunoblotting
Protein was extracted from infiltrated leaves using Plant Protein Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd) (Tang et al., 2023).Total proteins were separated by 12.5% SDS-PAGE (Epizyme), and the protein samples were transferred to a polyvinylidene difluoride (PVDF) membrane.Primary monoclonal antibodies (Proteintech) were diluted to 1:500 while horseradish peroxidase-conjugated goat anti-mouse IgG was used as a secondary antibody at a dilution of 1:10,000.

| Nucleotide extraction and detection analysis
Total DNA was extracted from diseased-citrus midribs (0.1 g) using Biospin Omini Plant Genomic DNA Extraction Kit (BioFlux), following the manufacturer's instruction.Quantification of CLas was performed using qPCR assay.CLas housekeeping gene CLasgyrA was detected, and 18S rRNA of C. sinensis was used as the endogenous control.BlastTaq 2× qPCR MasterMix (ABM) was used for qPCR amplification.The experiment contained three independent biological replicates, and three technical repeats were performed.
TaqMan qPCR was applied to quantify bacterial titre in citrus rootlets.DNA was extracted from hairy roots at 3 months after transformation, and the concentrations were adjusted to 10 ng/μL.The PCR system consisted of 16S rDNA primer HLBasr/ HLBasf, HLBp, DNA template, and TaqProbe 2× qPCR (ABM) in a total volume of 20 μL.The experiment contained four independent biological replicates, and three technical repeats were performed.The bacterial titres (CLas cell per μg of citrus DNA) were quantified with qPCR assay as was described by Li et al. (2006): CLas titre = 10 (12.715−Ct16S rRNA ×0.3264) ∕ 0.01.
Total RNA was extracted from citrus leaves using RNAiso Plus (Takara).In a 20-μL volume, 1 μg of total RNA was reverse transcribed with All-In-One 5× RT MasterMix (ABM) following the manufacturer's instructions.RT-qPCR assays were conducted to analyse gene transcript level.C. sinensis housekeeping gene CsGAPDH was used as the internal reference.The reaction was performed in a 10-μL volume.The experiments contained three independent biological replicates, and three technical repeats were performed.The 2 −ΔΔCt method was used for the determination of relative gene transcription (Livak & Schmittgen, 2001).

| Plant transformation and CLas inoculation
To generate CLas0185 transgenic citrus plants, the recombinant plasmid was transferred into A. tumefaciens EHA105.Agrobacteriummediated transformation of etiolated epicotyl segments of Wanjincheng was as previously described (Peng et al., 2015).
determined H 2 O 2 content in leaves of CsMsrB1-overexpressing/ silenced citrus plants.Its expression was manipulated through homologous transient overexpression and citrus leaf blotch virus (CLBV)-mediated virus-induced gene silencing (VIGS) assay in C. sinensis.CsMsrB1 overexpression was validated by immunoblotting at 3 dpi, and the fragment insertion was determined with RT-PCR at 1 mpi (Figure S4a,b).The transcript levels of CsMsrB1 were examined with RT-qPCR (Figure S4c,d).The overexpression of CsMsrB1 resulted in F I G U R E 2 CLas0185 impairs immune responses in Citrus sinensis.(a, b) Comparison of CLas0185 expression between psyllids and citrus (a), within a/symptomatic citrus leaves and rootlets (b).RNA was extracted from psyllid colonies and citrus plants that were infected by 'Candidatus Liberibacter asiaticus' (CLas), and tissue samples were collected from the same citrus plants.Transcript levels of CLas0185 were determined by reverse transcription-quantitative PCR (RT-qPCR) with CLasgryA (GenBank no.CP001677.5)as the internal reference.(c) Quantitative analysis of CLas growth during a 4-month period after graft inoculation in CLas0185-transgenic citrus lines (0185-OE) and wild-type (WT) group.C. sinensis 'Wanjincheng' was used for the generation of transgenic citrus.WT Wanjincheng plants of the same age were used as controls.mpi = months post-inoculation.The bacterial populations (CLas cells per μg of citrus DNA) were determined using quantitative PCR.(d) H 2 O 2 accumulation in leaves of 0185-OEs and WTs.The differences were determined by Student's t test, and different letters indicate statistically differences (p < 0.05).(e-h) Relative expression levels of pathogenesis-associated genes (PRs) and phloem protein 2 gene (PP2) in citrus plants.Transcript levels measured with RT-qPCR were normalized to levels in WTs using the CsGAPDH as endogenous control.Data in (a-h) represent mean ± SD.Asterisks indicate significant differences determined by Student's t test (*p < 0.05; **p < 0.01; ***p < 0.001; n = 3).The experiments comprised three independent biological replicates, and three technical repeats were performed.(i) CLas0185 attenuates huanglongbing symptom development.Images were taken at 0 and 12 months post-inoculation.Scale bar: 10 mm.F I G U R E 3 The interaction between CLas0185 and CsMsrB1 elevates CsMsrB1 abundance in Citrus sinensis and promotes host susceptibility to 'Candidatus Liberibacter asiaticus' (CLas).(a) CLas0185 binds with CsMsrB1 in a yeast two-hybrid (Y2H) assay.Yeast cotransformed with AD-T + BD-53 and AD-T + BD-Lam served as the positive and negative controls, respectively.Yeast co-transformed with AD-CsMsrB1 + BD-Lam and AD-T + BD-CLas0185 were auto-activation controls.Serial 10-fold dilutions of co-transformed yeast cells on double-dropout (DDO) and quadruple-dropout (QDO) + Xα-gal are shown.(b) Luciferase complementation imaging assay confirmed the interaction between CLas0185 and CsMsrB1 in the leaves of Nicotiana benthamiana at 3 days post-infiltration (dpi).The N-or C-terminal fragments of luciferase were fused to the C-terminus of CLas0185 and CsMsrB1.Co-expression of NbGAPC1-nLuc + NbATG3-cLuc was used as a positive control, and co-expression of CLas0185-nLuc + HA-cLuc or HA-nLuc + CsMsrB1-cLuc was applied as negative controls.(c) Glutathione-S-transferase (GST) pull-down assay demonstrated the interaction between CLas0185 and CsMsrB1.GST-CLas0185 and GST were expressed in Escherichia coli, immobilized on glutathione sepharose beads, and incubated with E. coli lysate containing His-CsMsrB1.Input and eluted (IP) proteins were immunoblotted using the anti-His and anti-GST antibodies.(d) CLas0185 increased the abundance of CsMsrB1.CsMsrB1 and GUS fused with FLAG were expressed in 0185-OE and wild-type (WT) groups through agro-infiltration.Proteins were extracted at 3 dpi, and the abundances of CsMsrB1 and β-glucuronidase (GUS) in 0185-OEs compared to those in WTs were determined with an anti-FLAG antibody.RuBisCO served as the endogenous control.Band intensity in blots was calculated by ImageJ based on three biological replicates, and the relative intensity was calculated.The experiment contained three independent biological replicates.(e, f) CLas titres in CsMsrB1-overexpressing (CsMsrB1-OE) and -silenced (CsMsrB1-RNAi) 0185-OEs.Agrobacterium rhizogenes-mediated hairy root genetic transformation was conducted to manipulate CsMsrB1 expression in CLas-infected 0185-OEs.Bacterial growth was detected at 3 months post-inoculation using TaqMan quantitative PCR.The experiment contained four independent biological replicates, and three technical repeats were performed.(g) Co-expression of CLas0185 and CsMsrB1 reduces H 2 O 2 accumulation.The H 2 O 2 content was measured in hairy roots of CsMsrB1-OEs and CsMsrB1-RNAis in a 0185-OE background.0185-OE served as the control.Data in (d-g) represent mean ± SD.Asterisks indicate significant differences determined by Student's t test (*p < 0.05; **p < 0.01; ***p < 0.001; ns, no significance).The experiments were performed twice, with similar results.asignificant decrease in basal H 2 O 2 levels in citrus leaves, while its silencing enhanced H 2 O 2 accumulation (Figure4a).Our findings provide direct evidence that CsMsrB1 is involved in scavenging H 2 O 2 .
Recombinant proteins were prepared to determine the effect of CsMsrB1 on CsAPX1 activity.CsAPX1 oxidized by H 2 O 2 has a higher molecular weight than the unoxidized (native) form.Added with CsMsrB1, oxidized CsAPX1 (O-CsAPX1) could be reduced, and the band in SDS-PAGE shifted back to the position of the unoxidised CsAPX1 (Figure 4f).Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that the percentage of Met36-O increased significantly in O-CsAPX1, while it decreased in substrates added with CsMsrB1 (Figure

F
CsAPX1 promotes 'Candidatus Liberibacter asiaticus' (CLas) proliferation.(a, b) CLas titres in CsAPX1-OE/RNAi lines.Agrobacterium rhizogenes-mediated hairy root genetic transformation was conducted to manipulate CsAPX1 expression in huanglongbingdiseased citrus plants.Bacterial growth was detected at 3 months post-inoculation (mpi) using TaqMan quantitative PCR.The experiment contained four independent biological replicates, and three technical repeats were performed.(c-e) Detection of ascorbate peroxidase (APX) activity.APX activity was detected in citrus plants expressing CLas0185 and CsMsrB1, as well as in rootlets co-expressing CLas0185 and CsMsrB1.Wild type (WT) served as the control for 0185-OE.GUS-OE and CLBV:GUS were used as controls for CsMsrB1-OE and CLBV:CsMsrB1, respectively.0185-OE served as the control for 0185-OE + CsMsrB1-OE and 0185-OE + CsMsrB1-RNAi, respectively.In (c), the differences were determined by Student's t test, and letters represent the difference between Mock and CLas inoculation (p < 0.05; n = 3).Data in (a-e) represent mean ± SD.Asterisks indicate significant differences determined by Student's t test (*p < 0.05; **p < 0.01; ***p < 0.001; n = 3).The experiments were performed twice, with similar results.(f) A schematic diagram illustrating that CLas0185 depresses plant immunity through targeting CsMsrB1.Perceived by hosts, CLas induces plant defence responses, including the reactive oxygen species (ROS) burst.To suppress plant immunity, CLas effector CLas0185 is delivered, which targets CsMsrB1 and increases its abundance.The latter restores the activity of CsAPX1, which scavenges H 2 O 2 accumulation, which negatively regulates plant defence.CC, companion cell; SE, sieve element.

CLas0185,
with the activity to suppress elicitor-triggered cell death, we discovered CsMsrB1 as its binding protein in C. sinensis, and their interaction facilitated CLas infection.On the one hand, CsMsrB1, restoring the enzymatic activity of CsAPX1, scavenges H 2 O 2 accumulation induced by CLas infection, which prevents ROS toxicity; on the other hand, it catalyses the reduction of Met-O back to Met, which may impair ROS-mediated plant defence.This study unveils the virulence strategy used by CLas0185 to depress host immunity.

4. 1 |
Plants, ACPs and growth conditions N. benthamiana plants were grown in a greenhouse at 25°C with an 18 h light/6 h dark cycle.The citrus plants were cultured at 28°C.CLas-infected mandarin orange plants (Citrus reticulata) were used for nucleotide extraction, as well as the source for graft inoculation.The epicotyl segments of C. sinensis 'Wanjincheng' were used for generation of transgenic citrus plants, and 1-year-old rough lemon plants (Citrus jambhiri) as rootstocks.cDNA of infected ACPs was from the National Navel Orange Engineering Research Center, Gannan Normal University, Ganzhou.

4. 2 |
Plasmid constructionGenomic DNA was extracted from CLas-infected C. reticulata plants; mature form of CLas0185 (a 150 bp coding region without the signal peptide) was amplified.The GUS fragment (a 300 bp coding region) was amplified from vector pLGN.Total RNA was extracted from leaves of C. sinensis, and reverse transcribed into cDNA, from which full-length of CsMsrB1 (XM_006477914.4) and CsAPX1 (XM_006488132.4) were amplified.For Agrobacterium-mediated transient expression in N. benthamiana, the coding sequences of CLas0185, CLas0185-GUS, and BAX were amplified with PCR or overlap PCR, and cloned into PVX, PVX-HA, and PVX-FLAG, respectively.PVX-HA, PVX-FLAG, PVX-GFP-HA, and PVX-CLas4425-FLAG were constructed in the laboratory.For the Y2H assay, CLas0185 and genes encoding enzymatic antioxidants, including CsAPXs, were cloned into the bait vector pGBKT7-BD (Clonetech).CsMsrB1 was inserted into the prey vector pGADT7-AD (Clonetech) to generate AD-CsMsrB1.For the LCI assay, the plasmids of CLas0185-nLuc, CsAPX1-nLuc, HA-nLuc, CsMsrB1-cLuc, and HA-cLuc were constructed.NbGAPC-nLuc and NbATG3-cLuc were constructed in the laboratory.For the pull-down assay, the plasmids of GST-CLas0185, GST-CsAPX1, and His-CsMsrB1 were generated.For Agrobacterium-mediated transient expression in C. sinensis, the coding sequences of CsAPX1 and CsMsrB1 were cloned into pLGN-HA and pLGN-FLAG, respectively.pLGN-GUS-HA and pLGN-GUS-FLAG were constructed.pLGN-HA and pLGN-FLAG were constructed in the laboratory.To prepare the stable overexpression plasmids, the amplified fragments of CLas0185, CsMsrB1, and CsAPX1 were cloned into pLGN.To obtain RNA interference plasmids, 300-bp fragments in CsMsrB1 and CsAPX1 were selected and inserted into the pGN vector between the AscI and SwaI restriction sites in the antisense orientation and between the BamHӀ and SalӀ restriction sites in the sense orientation to generation a hairpin.For the virus-induced gene silencing (VIGS) assay, the 300-bp fragments in CsMsrB1, CsAPX1, and GUS were cloned into pCLBV.
Transgenic lines were confirmed by GUS staining, and the buds without transformation served as the WT control.They were micrografted onto 1-month-old Wanjincheng rootstock seedlings.The resulting plantlets were further grafted onto C. jambhiri rootstock in a greenhouse.Transgenic plants were validated by PCR and RT-qPCR at